The present invention relates to a method for the production of recombinant viruses. It also relates to constructs used for carrying out this method, the producing cells, and the viruses thus produced. These viruses can be used as vector for the cloning and/or expression of genes in vitro, ex vivo or in vivo.
Vectors of viral origin are widely used for the cloning, transfer and expression of genes in vitro (for the production of recombinant proteins, for carrying out screening tests, for studying the regulation of genes and the like), ex vivo or in vivo (for the creation of animal models, or in therapeutic approaches). Among these viruses, there may be mentioned in particular adenoviruses, adeno-associated viruses (AAV), retroviruses, herpesviruses or vaccinia viruses.
The Adenoviridae family is widely distributed in mammals and birds and comprises more than one hundred different serotypes of nonenveloped double-stranded DNA viruses possessing a capsid of icosahedral symmetry (Horwitz, In: Fields B N, Knipe D M, Howley P M, ed. Virology. Third edition ed. Philadelphia: Raven Publishers, 1996: 2149-2171). In addition to its safety, the adenovirus has a very broad cellular tropism. Unlike the retrovirus, whose cycle is dependent on cell division, it can infect actively dividing cells such as quiescent cells and its genome is maintained in episomal form. Furthermore, it can be produced at high titres (1011 pfu/ml). These major assets of it one makes a most preferred vector for the cloning and expression of heterologous genes. The group C adenoviruses, particularly types 2 and 5, as well as the CAV-2-type canine adenoviruses, whose molecular biology is best known, are the source of the vectors currently used.
The adenovirus has a linear genome of 36 kb, terminating at each of these ends with inverted terminal repeat (ITR) sequences of 103 bp comprising a replication origin as well as an encapsidation signal situated near the left ITR (Shenk, Adenoviridae: The Viruses and Their Replication. In: Fields B N, Knipe D M, Howley P M, ed. Virology. Philadelphia: Raven publishers, 1996: 2111-2148). Three families of genes are expressed during the viral cycle:
The immediate-early genes (E1, E2, E3 and E4) which are involved in the regulation of cellular genes allowing in particular the entry of the cell into the S phase (E1A) and the inhibition of apoptosis (E1B). They are also involved in the regulation of early or late viral genes at the level of the transcription, splicing or transport of the messenger RNAs (E1A, E2A, E4). They also play a role in replication and in escaping the immune response.
The delayed-early genes (pIX and IVa2) are linked to the regulation of transcription of the late genes (IVa2) or to the assembling of the virion (pIX).
The late genes (L1 to L5) are transcribed from the strong promoter (MLP). A primary transcript of 28 kb makes it possible to generate the transcripts corresponding to the various structural proteins (core, penton, hexon) and nonstructural proteins participating in the assembling and in the maturation of the viral particles, by alternative splicing and the use of 5 polyadenylation sites.
Adenoviral vectors have been used for the cloning and expression of genes in vitro (Gluzman et al., Cold Spring Harbor, N.Y. 11724, p. 187), for the creation of transgenic animals (WO95/22616), for the transfer of genes into cells ex vivo (WO95/14785; WO95/06120) or for the transfer of genes into cells in vivo (see in particular WO93/19191, WO94/24297, WO94/08026).
As regards the adeno-associated viruses (AAV), they are relatively small DNA viruses which become integrated into the genome of the cells which they infect, in a stable and relatively site-specific manner. They are capable of infecting a broad spectrum of cells, without inducing any effect on cell growth, morphology or differentiation. Moreover, they do not seem to be involved in pathologies in man. The genome of the AAVs has been cloned, sequenced and characterized. It comprises about 4700 bases and contains, at each end, an inverted terminal repeat (ITR) region of about 145 bases which serves as replication origin for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left part of the genome, which contains the rep gene involved in the viral replication and the expression of the viral genes; the right part of the genome, which contains the cap gene encoding the virus capsid proteins.
The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO91/18088; WO93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528).
As regards the retroviruses, they are integrative viruses which selectively infect dividing cells. They therefore constitute vectors of interest for cancer or restenosis applications for example. The genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant vectors and their use in vitro or in vivo has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP 178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.
For their use as recombinant vectors, various constructs derived from viruses have been prepared, incorporating various genes of interest. In each of these constructs, the viral genome was modified so as to make the virus incapable of autonomously replicating in the infected cell. Thus, the constructs described in the prior art are viruses which are defective for certain regions of their genome which are essential for replication. In particular, as regards adenoviruses, the first-generation constructs exhibit a deletion in/of the E1 region, which is essential for viral replication, at the level of which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Moreover, to enhance the properties of the vector, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a heat-sensitive point mutation was introduced into the ts125 mutant, making it possible to inactivate the 72 kDa DNA binding protein (DBP) encoded by the E2 region (Van der Vliet et al., 1975). Other vectors comprise a deletion of another region essential for the viral replication and/or propagation, the E4 region. Adenoviral vectors in which the E1 and E4 regions are deleted have highly reduced transcription background noise and viral gene expression. Such vectors have been described, for example, in applications WO94/28152, WO95/02697, WO96/22378. Moreover, vectors carrying a modification at the level of the IVa2 gene have also been described (WO96/10088). In addition, so-called xe2x80x9cminimum adenovirusxe2x80x9d or xe2x80x9cpseudo-adenovirusxe2x80x9d vectors (or alternatively Adxcex94) containing only the regions necessary in cis for the production of the virus (ITR and encapsidation sequences) and lacking any coding viral sequence have also been described (WO94/12649, WO94/28152, WO95/02697), although their production remains very difficult, as explained below.
As regards the AAVs, the vectors described generally lack the entire coding regions Rep and Cap, which are replaced by nucleic acids of interest.
In the recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, completely or in part, and replaced by a heterologous nucleic acid sequence of interest. Moreover, the recombinant retroviruses may comprise modifications at the level of the LTRs in order to suppress the transcriptional activity, as well as large encapsidation sequences, comprising part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639).
Given their defective character in relation to the replication, the production of these various recombinant viruses involves the possibility of transcomplementing the functions deleted from the genome. The transcomplementation is precisely the source of major difficulties for the production of these viruses, and in particular the provision of the transcomplementation functions.
Two approaches have been developed in this regard. The first is based on the construction of transcomplementing lines, that is to say encapsidation lines. The second is based on the use of helper adenoviruses or of helper plasmids.
Various encapsidation lines of defective viruses have been constructed. These lines are capable of producing the functions deficient in the viral vector. Generally, these lines comprise, integrated into their genome, the region(s) deleted from the viral genome (E1, E2 and/or E4 for example for the adenovirus; gag, pol and/or env for the retrovirus, rep and/or cap for the AAV).
One of the lines known for the production of defective adenoviruses is for example the line 293 into which part of the adenovirus genome has been integrated. More precisely, the line 293 is a human embryonic kidney cell line containing the left end (about 11-12%) of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, the encapsidation region, the E1 region, including E1a and E1b, the region encoding the protein pIX and part of the region encoding the protein pIVa2. This line is capable of transcomplementing recombinant adenoviruses defective for the E1 region, that is to say lacking all or part of the E1 region, and of producing viral stocks having high titres. This line is also capable of producing, at permissive temperature (32xc2x0 C.), virus stocks comprising, in addition, the heat-sensitive E2 mutation. Other cell lines capable of complementing the E1 region have been described, based in particular on human lung carcinoma cells A549 (WO94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). Moreover, lines capable of transcomplementing several functions in the adenovirus have also been described. In particular, there may be mentioned lines complementing the E1 and E4 regions (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al., Hum. Gen. Ther. 6 (1995) 1575) and lines complementing the E1 and E2 regions (WO94/28152, WO95/02697, WO95/27071).
Various lines have also been described for the production of defective retroviruses, generally capable of expressing the gag, pol and env genes. Such lines are, for example, the PA317 line (U.S. Pat. No. 4,861,719), the PsiCRIP line (WO90/02806), the GP+envAm-12 line (WO89/07150), the BOSC line (WO94/19478) and the like. To construct recombinant retroviruses comprising a nucleic acid of interest, a plasmid comprising in particular the LTRs, the encapsidation sequence and the said nucleic acid is constructed, and then used to transfect an encapsidation line as described above, capable of providing in trans the retroviral functions deficient in the plasmid. The recombinant retroviruses produced are then purified by conventional techniques.
The use of lines can however have certain disadvantages. Thus, it is difficult, expensive and restrictive at the industrial level to construct and to validate such lines. Indeed, these lines should be stable and compatible with industrial uses. Furthermore, the lines described hardly make it possible to avoid the production of replicative contaminant viruses (RCA). Moreover, these lines do not allow at the present time, in a satisfactory manner for an industrial use, very highly defective viral genomes, such as for example minimum adenoviruses as described above, to be transcomplemented. Indeed, the adenovirus has a genome organized into various transcription units whose spatiotemporal regulation is very complex. It has so far not been possible to carry out satisfactorily the transcomplementation of an adenovirus deleted of all the coding viral sequences by expressing each transcription unit separately, in a constitutive or conditional manner, using a cell line. Thus, only a small proportion of the genome corresponding to the E1, E4 and pIX regions, and to the three proteins encoded by E2 (pol, DBP and p-TP) has been constitutively expressed using cell lines. The remainder of the genome corresponds to the major late transcription unit (MLTU) which produces all the messengers for the structural and nonstructural proteins from a primary transcript of 28 kb and is activated after replication of the genome. Now, to generate minimum adenoviruses, transcomplementation of these regions is essential. Neither do these lines make it possible to obtain very high recombinant retrovirus titres.
The second approach consists in cotransfecting with the defective viral genome a construct (plasmid or adenovirus) providing the complementation functions. In particular, the defective recombinant AAVs are generally prepared by cotransfection, in a cell line infected by a human helper virus (for example an adenovirus), of a plasmid containing a nucleic sequence of interest bordered by two AAV inverted terminal repeat (ITR) regions, and of a plasmid carrying the AAV complementation functions (rep and cap genes). Variants have been described in applications WO95/14771; WO95/13365; WO95/13392 or WO95/06743. The disadvantage of using a helper adenovirus lies mainly in the increased risks of recombination between the adenoviral vector and the helper adenovirus, and in the difficulty of separating the recombinant from the helper during the production and purification of the viral stocks. The disadvantage of using a helper plasmid, for example a plasmid Rep/cap, lies in the transfection levels obtained, which do not make it possible to produce high virus titres.
The present application now describes a new system for the production of viruses which makes it possible to overcome these disadvantages. The system of the invention is based on the use of a baculovirus to provide the complementation functions.
The production system according to the invention makes it possible, in a particularly advantageous manner, to dispense with the use of established complementation lines, to avoid the problems of RCA, and to transcomplement highly defective genomes. In addition, the system of the invention is applicable to any cell capable of being infected by the desired virus and by a baculovirus, and thus offers great flexibility of use.
A first subject of the invention therefore consists in a process for the production of defective recombinant viruses according to which the genome of the defective recombinant virus and a baculovirus comprising all or some of the functions necessary for the transcomplementation of the defective recombinant genome are introduced into a population of competent cells.
The process of the invention is therefore based on the use of a baculovirus to provide the complementing functions. Various approaches are possible. It is possible, first of all, to use competent cells not expressing any function of transcomplementation of the defective recombinant genome. In this case, it is possible to use either a baculovirus comprising all the functions necessary for the transcomplementation of the defective recombinant genome, or several baculoviruses each carrying one or more of the functions necessary for the transcomplementation of the defective recombinant genome. It is also possible to use a population of competent cells capable of already transcomplementing one or more functions of the defective recombinant genome (encapsidation line). In this case, the baculovirus(es) used will provide only the functions necessary for the transcomplementation of the defective recombinant genome which are not already transcomplemented by the competent cells.
As indicated above, the advantages of the system of the invention are numerous in terms of industrialization (no need for lines, no RCA, and the like), and in terms of applications (production of recombinant viruses carrying any type of deletion, and particularly of highly defective recombinant adenoviruses). In addition, since the baculovirus does not replicate in human cells, the viral preparation obtained is not contaminated by the baculovirus. Furthermore, the baculovirus being phylogenetically very distant from adenoviruses, there is no risk of recombination or transcomplementation between the two. This system therefore makes it possible, in an advantageous manner, to produce concentrated stocks of defective viruses, lacking RCA. This system is most particularly advantageous for the production of defective recombinant adenoviruses.
Baculoviruses are enveloped, circular double-stranded DNA viruses specific for invertebrates. Their prototype, AcNPV, has a genome of 133 kb. It is widely used as vector for the expression of eukaryotic genes in insect cells, starting from two strong promoters [polyhedrin (Ph) and P10], (King and Possee, The baculovirus expression system. London: Chapman and Hall, 1992.) AcNPV is capable of infecting some mammalian cells, but the genome is neither transcribed nor translated. Recently, Hofmann et al. (PNAS 92 (1995) 10099) have shown that in vitro, hepatocytic cells can be transduced by a purified recombinant baculovirus expressing the LacZ gene. No cellular toxicity was reported, even with a multiplicity of infection of 1000, and the transfection efficiency described in this article is about 50% for an MOI of 100.
The applicant has now shown that it is possible to infect various cell types with a recombinant baculovirus. In particular, the applicant has shown that it was possible, with a recombinant baculovirus, to infect cells of human origin such as immortalized embryonic cells. The applicant has also shown that it is possible to obtain a very high transduction efficiency ( greater than 80%). The applicant has also shown that it is possible to introduce, into a baculovirus, functions for complementation of an adenovirus, and to express these functions in a population of competent cells. The applicant has thus made it possible to show that the baculovirus constitutes an inert vector which can be advantageously used for the transfer and expression of virus complementation functions into mammalian, particularly human, cells. Other advantages of the system of the invention are in particular (i) the large cloning capacity which makes it possible to complement a whole adenoviral genome and (ii) the advanced development of the technology of the baculovirus.
The baculovirus carrying the functions for complementation of the virus is also designated in the text which follows helper baculovirus. It may comprise various functions for complementation of the virus.
Thus, the helper baculovirus may comprise the E1 region of the adenovirus. A Baculo-E1 can be used for the production of first-generation adenoviruses, that is to say adenoviruses defective for the E1 region (Adxcex94E1), regardless of its E3 status (i.e. defective Adxcex94E1, xcex94E3, or not). The production of first-generation defective recombinant adenoviruses (defective for the E1, and possibly E3, region) constitutes a first particularly advantageous application of the process of the invention. As indicated above, various lines have been described in the literature which are capable of transcomplementing the E1 function (cells 293, cells A549, cells 911, and the like). However, various zones of homology exist between the region carrying the transcomplementation functions which is integrated into the genome of the line and the DNA of the recombinant virus which it is desired to produce. Because of this, during production, various recombination events may occur, generating replicative viral particles, in particular type E1+ adenoviruses. This may be a single recombination event followed by breaking of the chromosome, or a double recombination. These two types of modification lead to reintegrating into its initial locus within the adenoviral genome the E1 region contained in the cellular genome. Moreover, given the high titres of recombinant vector which are produced by the line 293 (greater than 1012), the probability of these recombination events occurring is high. In fact, it has been observed that numerous batches of first-generation defective recombinant adenoviral. vectors were contaminated by replicative viral particles, which may constitute a major disadvantage for pharmaceutical uses. Indeed, the presence of such particles in therapeutic compositions would induce in vivo an uncontrolled viral propagation and dissemination with risks of inflammatory reaction, of recombination and the like. The contaminated batches cannot therefore be used in human therapy.
The present invention makes it possible to overcome these disadvantages. Indeed, according to one embodiment of the process of the invention, the genome of the recombinant adenovirus defective for the E1, and possibly E3, region is introduced into the competent cells, these cells are infected, simultaneously or otherwise, with a baculovirus comprising the E1 region, the adenovirus E1 region present in the baculovirus and the genome of the defective recombinant adenovirus comprising no zone of homology (overlapping) capable of giving rise to recombination. According to this embodiment, it is thus possible to rapidly produce, without an established line, stocks of first-generation recombinant adenoviruses free of RCA. Moreover, as indicated below, the stocks of recombinant adenoviruses thus generated, free of RCA, can be used as starting material for a new production, by coinfection in the competent cells with a baculovirus.
The helper baculovirus may also comprise the E2 region of the adenovirus, in full or in part, particularly the E2a and/or E2b region. A baculo-E2 may be used to produce, in competent cells, adenoviruses defective for the E2 region (Ad-xcex94E2), and possibly for the E3 region (Ad-xcex94E2, xcex94E3). In addition, in competent cells capable of complementing the E1 region of the adenovirus, the baculo-E2 may allow the production of recombinant adenoviruses defective for the E1 and E2 (Ad-xcex94E1, xcex94E2) and possibly E3 (Ad-xcex94E1, xcex94E2, xcex94E3) regions. Likewise, in competent cells capable of complementing the E1 and E4 regions of the adenovirus (for example in IGRP2 cells), the baculo-E2 may allow the production of recombinant adenoviruses defective for the E1, E2 and E4 (Ad-xcex94E1,xcex94E2,xcex94E4) and possibly E3 (Ad-xcex94E1,xcex94E2,xcex94E3,xcex94E4) regions.
The helper baculovirus may also comprise the E4 region (in full or in part) of the adenovirus. A baculo-E4 may be used to produce, in competent cells, adenoviruses defective for the E4 region (Ad-xcex94E4), and possibly for the E3 region (Ad-xcex94E4,xcex94E3). In addition, in competent cells capable of complementing the E1 region of the adenovirus, the baculo-E4 may allow the production of recombinant adenoviruses defective for the E1 and E4 (Ad-xcex94E1,xcex94E4) and possibly E3 (Ad-xcex94E1,xcex94E4,xcex94E3) regions.
The helper baculovirus may also comprise the E1 and E4 regions (in full or in part) of the adenonvirus. A baculo-E1,E4 may be used to produce, in competent cells, adenoviruses defective for the E1 and E4 (Ad-xcex94E1,xcex94E4) and possibly E3 (Ad-xcex94E1,xcex94E4,xcex94E3) regions, as illustrated in FIG. 1.
In addition, to generate viruses defective for the E1 and E4 regions, it is also possible to use two helper baculoviruses, one expressing the E1 function, the other the E4 function, in full or in part.
In the same manner, the helper baculovirus may comprise the E1, E2 and E4 regions (in full or in part), and possibly the regions carrying the late genes (L1-L5).
The helper baculovirus may also comprise the AAV R ep and/or Cap regions. A baculo-Rep/Cap thus makes it possible to complement, in a line of competent cells, an AAV genome lacking any coding viral sequence (FIG. 5).
The baculovirus may also comprise the gag, pol and/or env regions of a retrovirus. A baculo-gag/pol/env thus makes it possible to complement, in a line of competent cells, a retroviral genome lacking any coding viral sequence.
It is also possible to use a baculovirus comprising the gag/pol regions and a second baculovirus containing the env region.
In general, it is preferable that the genome of the defective recombinant virus and the complementatibn regions present in the baculovirus do not overlap. This makes it possible, indeed, to avoid the risks of recombination and thus the generation of RCA. This is particularly important for the generation of first-generation adenoviruses (Ad-xcex94E1). In this case, the E1 region introduced into the baculovirus is defined so that it does not possess any common sequence with the recombinant genome. To do this, it is possible, for example, to delete from the recombinant genome a region larger than the complementing region inserted into the baculovirus, as illustrated in the examples. This is also advantageous for the generation of Ad-xcex94E1,xcex94E4 adenovirus.
Thus, in a specific embodiment of the process of the invention, the genome of the defective recombinant virus is introduced into the competent cells, these cells are infected, simultaneously or otherwise, with a baculovirus comprising all or some of the functions necessary for the complementation of the defective genome, the complementation functions present in the baculovirus and the genome of the defective recombinant virus comprising no zone of homology capable of giving rise to recombination. Advantageously, the viral genome is a recombinant adenovirus genome defective for the E1 region and the baculovirus carries a region of the adenovirus capable of transcomplementing the E1 region. According to another variant, the viral genome is a recombinant adenovirus genome defective for the E1 and E4 regions and the baculovirus carries two adenovirus regions capable of transcomplementing the said regions or two baculoviruses are used, one carrying a region of the adenovirus capable of transcomplementing the E1 region and the other a region of the adenovirus capable of transcomplementing the E4 region, without zones of homology with the defective adenoviral genome.
According to a specific embodiment, all the coding regions of the adenovirus are carried by one or more helper baculoviruses. According to a more specific embodiment, only one helper baculovirus comprising all the coding regions of the adenovirus is used. Such a helper baculovirus can thus be used to transcomplement minimum recombinant adenoviruses. Such a baculovirus may in particular comprise the whole of one adenoviral genome, with the exception of the encapsidation region and possibly the ITRs, as illustrated in the examples.
Preparation of the Complementation Functions
The complementation functions introduced into the helper baculovirus may be derived from viruses of different serotypes.
As regards adenoviruses, various serotypes exist whose structure and properties vary somewhat, but which exhibit a comparable genetic organization. More particularly, the complementation functions used for the construction of the baculoviruses according to the invention are derived from an adenovirus of human or animal origin.
As regards adenoviruses of human origin, there may be mentioned, preferably, those classified in the C group. Still more preferably, among the various human adenovirus serotypes, the use of the type 2 or 5 adenoviruses (Ad2 or Ad5) is preferred within the framework of the present invention. It is also possible to use regions derived from type 7 or 12 adenoviruses, belonging to groups A and B. Among the various adenoviruses of animal origin, the use of the adenoviruses of canine origin, and particularly all the strains of the CAV2 adenoviruses [manhattan or A26/61 strain (ATCC VR-800) for example] is preferred within the framework of the invention. Other adenoviruses of animal origin are mentioned in particular in application WO94/26914 incorporated into the present by reference.
In a preferred embodiment of the invention, the complementation function is derived from a group C human adenovirus genome. More preferably, it is derived from the genome of an Ad2 or Ad5 adenovirus.
The regions carrying the various complementation functions may be obtained, from an adenoviral genome, by enzymatic cleavages according to methods known to persons skilled in the art. These regions may optionally be modified in order to reduce their size, or to replace certain regulatory elements (promoter, enhancer and the like) with heterologous elements. In general, these regions are prepared as follows: the DNA of an adenovirus is purified by caesium chloride gradient centrifugation or obtained in vitro from a prokaryotic (WO96/25506) or eukaryotic (WO95/03400) plasmid. The DNA is then cleaved with appropriate restriction enzymes and the fragments obtained, carrying the desired complementation functions, are identified and selected. The choice of the restriction enzymes used depends on the desired complementation functions. It is then guided by the restriction maps and the published sequences of the adenoviral genomes. Thus, the E1 region may be isolated in the form of fragments carrying all the reading frames of E1A and E1B downstream of the E1A promoter. The E4 region may be isolated in the form of fragments carrying the whole of the reading frames, or only part of them, and preferably the frames ORF3 or ORF6 or ORF6-ORF6/7.
Similar methodologies are used to prepare the AAV and recombinant retrovirus complementation regions. Thus, the AAV rep and/or cap regions may be obtained by enzymatic cleavage from the viral DNA isolated from various AAV serotypes. This is preferably AAV-2. For retroviruses, the gag, pol and/or env regions may also be obtained according to conventional molecular biological techniques, from various types of retroviruses, such as in particular MoMuLV (Murine Moloney Leukaemia Virus; also called MOMLV), MSV (Murine Moloney Sarcoma Virus), HaSV (Harvey Sarcoma Virus); SNV (Spleen Necrosis Virus), RSV (Rous Sarcoma Virus) or Friend""s virus.
Construction of the Helper Baculovirus
The fragments carrying the complementation regions are then subcloned into a plasmid vector allowing their manipulation (finer digestions, PCR, additions of regulatory sequences, and the like), for example in a prokaryotic or eukaryotic organism. The final fragment obtained, encoding the complementation function(s) is then introduced into the helper baculovirus using conventional molecular biological techniques. Specifically, the fragment is cloned between two sequences homologous to a region of the genome of a baculovirus, and then the resulting fragment or plasmid is cotransfected with the genome of a baculovirus into insect cells (conventionally Sf9 and Sf21, spodoptera frugiperda cells, but also Tn-368 and High-Five(trademark) BTI-TN-5B1-4 (Gibco), trichopulsia ni cells, or any other insect cell permissive to baculoviruses and capable of being used for their production). The homologous recombination between the plasmid or fragment and the genome of the baculovirus generates the desired recombinant baculovirus, which may be recovered and purified according to conventional methods (see in particular King and Possee: the baculovirus expression system. London: Chapman and Hall, 1992). For the construction of the recombinant baculoviruses, various kits comprising shuttle vectors are commercialized and may be used according to the recommendations of the manufacturers. In particular, it is possible to use the shuttle vectors pBAC marketed by the company Clontech, the vectors pAc (Verne et al., Bio/Technology 6 (1988) 47, Pharmingen, USA), the vectors pBlue-Bac (Invitrogen) or the vectors pBSV (Boehringer). The complementation functions may thus be inserted into different sites of the baculovirus, and in particular into the locus of the polyhedrin gene or of the p10 gene. Moreover, various baculovirus strains can be used, such as in particular AcNPV or Bombyx mori (Maeda et al., Nature 315 (1988) 592). Furthermore, the baculovirus used may be modified to enhance/change its tropism. It is indeed possible to modulate the tropism of the viral vectors by modifying their surface proteins so as (i) to limit it by fusion of the viral proteins with a specific ligand (light immunoglobulin chain, Gastrin-Releasing Peptide) or (ii) to broaden it by formation of pseudotypes with a heterologous viral glycoprotein [G of the Vesicular Stomatitis Virus (VSV)], [Liu et al., J. Virol 70(4) (1996) 2497; Michael et al., Gene Ther. 2 (1995) 660]. Recently, it was shown that the baculovirus surface glycoprotein (gp64) fused with gp120 of the HIV virus was capable of binding to the CD4 receptor (Boublik et al. Bio/Technology 13 (1995) 1079). This modification of gp64 does not affect the viability of the baculovirus in insect cells. A similar construct with the G of VSV should make it possible to enhance the tropism of the baculovirus for mammalian cells and therefore to increase the transduction efficiency of the Huh7 cells as well as other cell lines.
In the helper baculovirus, the complementation functions are advantageously placed under the control of a heterologous promoter (i.e. of a different origin from the baculovirus), which is functional in competent cells. It appears, indeed, that the baculovirus promoters do not make it possible to obtain sufficient levels of expression of the complementation functions in cells other than insect cells, and are therefore not the most appropriate for the applications of the invention. The promoter may first of all be the actual promoter (homologous promoter) of the complementation functions of the virus (E1A, E4, E2, MLP promoter for the adenovirus, P5 or P19 promoters of AAV, the LTR promoter of RSV, and the like). It may also be any promoter of different origin which is functional in the competent cell used. To this effect, there may be mentioned for example the promoters of genes expressed in this cell, or known ubiquitous promoters such as for example the promoter of the PGK gene, the immediate-early promoter of CMV, the promoter of the TK gene of the herpesvirus or alternatively the LTR promoter of RSV. It may also be a regulated promoter, such as for example the promoter of the MMTV virus, a promoter responding to hormones, for example of the GRE5 type, or a promoter regulated by tetracycline (WO). Advantageously, it is an inducible or strong ubiquitous, homologous promoter.
Thus, another subject of the present invention relates to a recombinant baculovirus comprising, inserted into its genome, a nucleic acid encoding a complementation function of a virus placed under the control of a heterologous promoter. More particularly, the complementation function is a protein necessary for the production of the said virus, and whose coding region is inactive (mutated, deleted and the like) in the defective viral genome. For adenoviruses, the complementation function is more particularly chosen from all or some of the functions encoded by the E1, E2, E4, L1-L5, pIX and IVa2 regions of the adenovirus, alone or in combination. For the AAV, they are functions encoded by the Rep and/or Cap regions; and for the retrovirus, gag, pol and/or env. Advantageously, the nucleic acid corresponds to a region of a viral genome comprising the region encoding the complementation function chosen. In particular, it is a fragment of a genome of adenoviruses of serotype Ad2 or Ad5, MoMLV or AAV-2. In a particularly preferred manner, the nucleic acid also comprises the promoter region which is naturally responsible for the expression of the complementation functions chosen.
By way of a specific example, the present invention relates to a baculovirus comprising all or part of the E1 region of an adenovirus. More particularly, it is a baculovirus comprising the E1a, E1b or E1a and E1b region. The E1 region of the adenovirus is located at the level of nucleotides 104 (promoter E1a) to 4070 (polyA E1b) of Ad5. In particular, the TATA box of the E1a promoter is located at the level of nucleotide 470, the ATG codon of E1a at the level of nucleotide 560, and the stop codon E1b at the level of nucleotide 3511. There may be mentioned by way of precise example a baculovirus comprising a fragment 391-3511. This helper baculovirus is particularly suitable for the production of recombinant adenoviruses defective for the E1 region, carrying a larger deletion than this 391-3511 fragment. In particular, it is suitable for the production of first-generation adenoviruses, without RCA, carrying a deletion in the E1 region covering nucleotides 383-3512 inclusive.
Another specific example of a baculovirus according to the invention comprises, for example, all or some of the E1 and E4 regions of the adenovirus. The E4 region of the adenovirus consists of 7 open reading frames, designated ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7. Among the proteins encoded by these various ORFs, those produced by ORF3 and ORF6 appear to allow the xe2x80x9creplicationxe2x80x9d of the virus, and therefore the transcomplementation of an adenovirus defective for the E4 region, even in its entirety. As a result, the helper baculovirus of the invention advantageously comprises all the E4 region or only part thereof comprising at least the ORF3 or ORF6 frame. The various parts of the E4 region may be obtained by enzymatic cleavages or modified according to methods known to persons skilled in the art. In particular, the reading frame ORF6 may be isolated from the E4 region in the form of a BglII-PvuII fragment, corresponding to nucleotides 34115-33126, and the reading frames ORF6-ORF6/7 may be isolated from the E4 region in the form of a BglII-BglII fragment corresponding to nucleotides 34115-32490 of the genome of Ad5. The baculovirus may also comprise the whole of the reading frames ORF1-ORF7 (for example in the form of a 32800-35826 or 32811-35614 or 32811-35640 fragment). It is understood that other fragments may be determined on the basis of published sequences of the adenoviral genomes. The use of a baculovirus carrying a reduced unit of the E4 region is advantageous because it allows the transcomplementation of a defective adenoviral genome carrying a larger deletion of the E4 region, therefore without a zone of homology, and thus to avoid any possibility of recombination.
According to a first embodiment, the nucleic acid encoding the complementation function(s) is introduced into the helper baculovirus in the form of an expression cassette. This embodiment is the easiest to use. It is particularly suitable for the production of recombinant adenoviruses defective for immediate-early genes and for the production of defective recombinant AAVs and retroviruses.
According to another embodiment, the nucleic acid encoding the complementation function(s) is introduced into the helper baculovirus in the form of an excisable cassette, generating a replicative molecule in the competent cell. The replication of the cassette in the cell makes it possible advantageously to increase the copy number of the complementing genes, and thus to enhance the production levels of the system. This embodiment is particularly suitable for the production of very highly defective recombinant adenoviruses, particularly defective for the structural genes. In particular, this embodiment is particularly suitable for the production of xe2x80x9cminimumxe2x80x9d adenoviruses. Indeed, the quantity of structural protein is a limiting factor for the production of high titres of highly defective adenoviruses (minimum adenovirus type). This embodiment makes it possible, for the first time, to considerably increase the intracellular levels of transcomplementing proteins, particularly of structural proteins of the adenovirus (encoded by the L1 to L5 regions), up to levels compatible with the transcomplementation of minimum adenoviruses.
Thus, the applicant has shown that it is possible to construct recombinant baculoviruses comprising a heterologous region capable of being excised in a cell, preferably in an inducible and regulated manner, in order to generate a circular and replicative molecule (of episomal type).
The excision is advantageously carried out by a site-specific recombination mechanism, and the replication in the cell is brought about by a replication origin, independent of the state of cell division.
More preferably, the site-specific recombination used according to the process of the invention is obtained by means of two specific sequences which are capable of recombining with each other in the presence of a specific protein, generally called recombinase. These specific sequences, arranged in the appropriate orientation, flank in the baculovirus the sequences encoding the complementation functions. Thus, the subject of the invention is also a recombinant baculovirus comprising, inserted into its genome, at least one DNA region flanked by two sequences allowing a site-specific recombination and positioned in direct orientation, the said DNA region comprising at least one replication origin functional in competent cells and a nucleic acid encoding a complementation function of a virus.
The sequences allowing the recombination which are used in the framework of the invention generally comprise from 5 to 100 base pairs, and more preferably less than 50 base pairs. They may belong to different structural classes, and in particular to the family of the recombinase of the P1 bacteriophage or of the resolvase of a transposon.
Among the recombinases belonging to the bacteriophage 1 integrase family, there may be mentioned in particular the integrase of phages lambda (Landy et al., Science 197 (1977) 1147), P22 and F80 (Leong et al., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenzae (Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase of the P1 phage, the integrase of the plasmid pSAM2 (EP 350 341) or the FLP recombinase of the plasmid 2 xcexcm of the yeast Saccharomyces cerevisiae. 
Among the recombinases belonging to the Tn3 transposon family, there may be mentioned in particular the resolvase of the Tn3 transposon or of the gd, Tn21 and Tn522 transposons (Stark et al., 1992); the Gin invertase of the mu bacteriophage or the resolvase of plasmids, such as that of the fragment par of RP4 (Abert et al., Mol. Microbiol. 12 (1994) 131).
According to a preferred embodiment, in the recombinant baculoviruses of the present invention, the sequences allowing the site-specific recombination are derived from a bacteriophage. More preferably, they are sequences for attachment (attp and attB sequences) of a bacteriophage or of derived sequences. These sequences are capable of specifically recombining with each other in the presence of a recombinase called integrase. By way of specific examples, there may be mentioned in particular the sequences for attachment of the phages lambda, P22, F80, P1, HP1 of Haemophilus influenzae or of the plasmid pSAM2, or 2 xcexcm.
Still more preferably, the sequences allowing the site-specific recombination are represented by the recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which specifically recognizes a nucleotide sequence of 34 base pairs called lox P site (Sternberg et al., J. Mol. Biol. 150 (1981) 467). This sequence is composed of two palindromic sequences of 13 bp separated by a conserved sequence of 8 bp. The site-specific recombination is advantageously obtained using LoxP sequences or derived sequences, and the Cre recombinanse.
The term derived sequence includes the sequences obtained by modification(s) of the recombination sequences above, conserving the capacity to specifically recombine in the presence of the appropriate recombinase. Thus, it may involve reduced fragments of these sequences or on the contrary fragments extended by addition of other sequences (restriction sites and the like). It may also involve variants obtained by mutation(s), particularly by point mutation(s).
According to a preferred embodiment of the invention, the sequences allowing a site-specific recombination are therefore LoxP sequences of the P1 bacteriophage, and the recombination is obtained in the presence of the Cre protein. In this regard, the recombination may be obtained by bringing the competent cells directly into contact with the Cre recombinase, or by expression of the gene encoding the Cre recombinase in the competent cells. Advantageously, the Cre recombinase is produced in the cell by inducing the expression of the corresponding gene. Thus, the gene encoding the recombinase is advantageously placed under the control of an inducible promoter, or constructed in a regulatable form. In this regard, there is advantageously used a fusion between Cre and the steroid hormone (oestradiol, progesterone and the like) binding domain allowing the activity of Cre to be regulated and therefore the recombination event to be induced (Metzger et al., PNAS 92 (1995) 6991). More generally, the expression of the recombinase may be controlled by any strong promoter, regulated or otherwise. The expression cassette may be transfected into the competent cells, or integrated into the genome of the competent cells, as illustrated in the examples.
This system therefore makes it possible to generate replicative molecules producing, in the competent cells, high levels of virus, particularly adenovirus, complementation function. This type of construct is particularly suitable for the complementation of highly defective genomes, in particular of adenoviral genomes defective for the late genes. Thus, a specific construct according to the invention is represented by a baculovirus comprising, inserted into its genome, at least a DNA region flanked by two LoxP sequences positioned in direct orientation, the said DNA region comprising at least one replication origin functional in the competent cells and one nucleic acid encoding a complementation function of an adenovirus. Advantageously, the complementation functions comprise all or some of the immediate-early genes present in the E1, E2 and E4 regions. Still more preferably, the complementation functions comprise all or some of the immediate-early genes and of the delayed-early genes. Preferably, the complementation functions allow the complementation of a recombinant adenovirus lacking any coding viral sequence. In particular, the complementation functions consist of the whole of the adenoviral genome, with the exception of the ITRs and of the packaging region. According to a specific variant, the complementation functions consist of a complete adenoviral genome lacking, however, the packaging region (Ad.Psi-). This genome comprises in particular the ITRs which serve for the replication of the genome in the competent cells, after excision.
To bring about the replication of the episomal molecule, the latter therefore contains a replication origin functional in the competent cells used. This replication origin preferably consists of the actual ITR sequences of the adenovirus, which allow substantial amplification of the molecule. It may also be another replication origin allowing, preferably, amplification by a factor greater than 20 of the viral DNA in the competent cell. There may be mentioned, by way of illustration, the origin OriP/EBNA1 of the EBV virus or the E2 region of the papilloma virus. It is understood that the ITR sequences of the adenovirus constitute a preferred embodiment.
For carrying out the process of the invention, the helper baculovirus(es) are generally used at a Multiplicity of Infection (MOI) allowing a large population of cells to be infected, without significantly impairing cell viability. Generally, it is more particularly between 10 and 1000. The MOI corresponds to the number of viral particles per cell. The MOI may be easily adjusted by persons skilled in the art depending on the competent cells used, essentially on the basis of two criteria: the infection efficiency and the possible toxicity. Advantageously, the MOI used for the helper baculovirus is between 20 and 500.
Introduction of the Viral Genome
As indicated above, the process of the invention comprises the introduction, into competent cells, of the helper baculovirus and of the recombinant viral genome. In this regard, the genome of the defective recombinant adenovirus may be introduced in various ways into the competent cell.
It may, first of all, be a purified defective recombinant adenovirus, advantageously free of RCA. In this case, the competent cells are infected with the defective recombinant adenovirus and with the helper baculovirus. The infection with the recombinant adenovirus makes it possible to introduce into the competent cell the corresponding genome, which is then amplified and encapsidated in order to produce stocks at a high titre, free of RCA. This embodiment is particularly advantageous for generating first-generation viruses (Ad-xcex94E1; Ad-xcex94E1, xcex94E3). Indeed, these viruses are difficult to produce at high titres, without contamination with RCAs. According to the process of the invention, it is now possible, starting with a first-generation defective recombinant adenovirus, by coinfection in a competent cell with a baculovirus comprising the E1 region, to obtain concentrated stocks, of high quality. This embodiment is also advantageous for the production of viruses defective in two or three essential regions of their genome (E1, E2, E4 in particular). In general, this embodiment is advantageous because the efficiency of infection with the adenovirus is very high (greater than the efficiency of transfection with DNA), and therefore makes it possible to generate concentrated stocks. In this embodiment, the recombinant adenovirus and the recombinant baculoviruses are used at multiplicities of infection (MOI) allowing a large population of cells to be infected, without significantly impairing cell viability. The MOI used for the baculovirus is that stated above (between 10 and 1000). As regards the adenoviruses, it is advantageously between 1 and 1000, preferably between 1 and 500, still more preferably between 1 and 100. The MOI used for the adenovirus is also adjusted according to the cell type chosen. The MOI range may be easily determined by persons skilled in the art using, for example, an adenovirus and a baculovirus comprising a separate marker gene, in order to measure the efficiency of infection and any competition. More preferably, the MOI of the adenovirus is less than 50, for example between 1 and 20.
According to another particularly advantageous embodiment, the genome of the defective recombinant adenovirus is introduced in the form of DNA. In this case, the genome is introduced by transfection, optionally in the presence of a transfection-facilitating agent (lipids, calcium phosphate and the like). The recombinant genome thus introduced may be prepared in vitro according to various techniques, and in particular in E. coli (WO96/25506) or in a yeast (WO95/03400). This embodiment is in particular useful for generating a first batch of defective recombinant virus, free of RCA, which can then in turn be used to produce stocks with a high titre according to the preceding embodiment.
The genome of the defective recombinant adenovirus may also be introduced using another recombinant baculovirus. According to this embodiment, the genome of the defective recombinant adenovirus is prepared in vitro, for example as indicated above, and then introduced into a baculovirus, in the form of a cassette capable of being excised in the competent cell. According to this embodiment, the competent cells are put in the presence of a baculovirus carrying the genome of the defective recombinant adenovirus, and of one or more helper baculoviruses (carrying the complementation functions). This embodiment is particularly advantageous for the production of highly defective recombinant adenoviruses. By virtue of this system, it is indeed possible to introduce into the population of competent cells high quantities both of the highly defective recombinant genome and of the corresponding complementation functions.
In this regard, a process of the invention therefore comprises the coinfection of competent cells with a baculovirus carrying the genome of the defective recombinant adenovirus, and one or more helper baculoviruses carrying the complementation functions. The MOI values used in this embodiment are also between 10 and 1000 for each of the baculoviruses used.
Two types of constructs have been prepared in the prior art for the production of minimum adenoviruses: (1) the transgene (xcex2-galactosidase) cloned between the ITRs, bordered by a unique restriction site or (2) the right and left ITRs cloned in direct orientation in 5xe2x80x2 of the transgene (Fisher et al., Virology 217 (1996) 11; Kumar-Singh et al., Hum. Mol. Genet. 5 (1996) 913). Minimum adenoviruses were produced in the cells 293 by transfection of linearized (1) or circular (2) DNA, the viral proteins necessary for the replication and for the encapsidation of the minigenome being provided in trans by a helper virus (Adxcex94E1). The minimum adenoviruses behave like interfering defective (ID) particles and are progressively amplified during successive passages. The major problem posed by the use of this methodology is the separation of the two types of particles produced, responsible for the contamination of the stocks by the helper virus, and the very low titres of minimum adenoviruses thus obtained (less than 108 pfu/ml).
The present application makes it possible, for the first time, to generate minimum adenoviruses using a baculovirus to deliver the adenoviral minigenome and a baculovirus to provide all the transcomplementation functions (complementing genome).
The recombinant adenoviral genome is advantageously introduced into the baculovirus, between two sequences allowing a site-specific recombination in the competent cells, as described for the helper baculovirus.
The present application describes in particular a system for the production of a minimum adenovirus using a baculovirus to deliver the adenoviral minigenome with the aid of the loxP/Cre system and a baculovirus to provide all the transcomplementation functions (complementing genome), also with the aid of a Cre/loxP system (see FIGS. 2-4).
According to another embodiment, the site-specific recombination system used to deliver the complementation functions is different from that used to deliver the genome of the recombinant adenovirus. In particular, the LoxP/Cre system may be used to deliver the defective adenoviral genome and the AttP/AttB system to deliver the complementation function(s).
The process of the invention thus makes it possible to construct an adenoviral vector deleted of all coding viral sequences and comprising only the ITRs and the encapsidation signal (minimum adenoviruses). This vector can theoretically accommodate up to 37 kb of exogenous sequence whereas the cloning capacity of current vectors does not exceed 8.5 kb. It thus makes it possible to clone genes of large size such as the dystrophin gene (14 kb) with all their regulatory elements (promoter, enhancer, introns and the like) so as to obtain an optimum expression, in the target tissue. Furthermore, the absence of any immunogenic viral sequence should increase the duration of expression of the transgene in quiescent tissues.
The genome of AAV or the defective retrovirus may also be introduced in the form of a virus, a genome or a plasmid, according to the techniques described above.
Competent Cells
The process of the invention may be carried out in various types of cells. For the purposes of the invention, xe2x80x9ccompetent cellxe2x80x9d is understood to mean a cell permissive to infection by the baculovirus and the virus to be produced, and allowing a productive viral cycle for the latter. The capacity to infect cells with these viruses can be determined using recombinant viruses expressing a marker gene such as the E. coli LacZ gene. It is preferably a mammalian cell, still more preferably a cell of human origin. The competent cells used may be quiescent cells or actively dividing cells, established lines or primary cultures. They are advantageously mammalian cells compatible with an industrial use, that is to say without a known pathogenic character, capable of being cultured and, where appropriate, of being stored under appropriate conditions. Advantageously, the cells used are hepatic, muscular, fibroblastic, embryonic, nerve, epithelial (pulmonary) or ocular (retinal) cells. There may be mentioned, by way of nonlimiting example, the cells 293 or any derived cell comprising an additional complementation function (293E4, 293E2a, and the like), the A549 cells, the HuH7 cells, the Hep3B cells, the HepG2 cells, the human retinoblastic cells (HER, 911), the HeLa cells, the 3T3 cells or the KB cells.
To carry out the process of the invention, the genome of the recombinant virus and the baculovirus may be introduced into the population of competent cells simultaneously or spaced out over time. Advantageously, the cells are brought into contact both with the recombinant genome and the helper baculovirus. In the case of a system generating replicative molecules in vivo, the recombinase is introduced or expressed beforehand, simultaneously or subsequently.
The production of the viruses generally leads to the lysis of the cells. The viruses produced can therefore be harvested after cell lysis, according to known purification methods. They can then be packaged in various ways depending on the desired use. Moreover, to avoid any risk of contamination of the viral stock with possible traces of baculoviruses that have not penetrated into the competent cells (helper baculovirus or baculovirus providing the recombinant viral genome), it is possible to apply the following techniques:
it is possible to purify the adenoviruses by chromatography according to the method described in application FR96/08164. This technique makes it possible to separate the adenovirus from any possible residual baculovirus;
it is also possible to cause organic solvents (for example ether, chloroform) to act on the stocks of purified adenovirus. Indeed, the baculovirus is an enveloped virus (glycoprotein envelope), and is therefore very sensitive to any organic solvent (which extracts the lipids from its envelope); in contrast, the adenovirus is not enveloped, and the same solvents have no effect on it;
it is also possible, by CsCl gradient purification, to separate, by density, any residual baculovirus and the recombinant virus.
These three methods can be used independently or conjointly. Moreover, any other method known to a person skilled in the art can also be used.
Use of the Viruses
The viruses thus produced can be used for the cloning, transfer and expression of genes in vitro, ex vivo or in vivo. Such genes of interest are, for example, genes encoding enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, and the like (FR 9203120), growth factors, neurotransmitters or their precursors of synthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, and the like, apolipo proteins: ApoAI, ApoAIV, ApoE, and the like (WO94/25073), dystrophin or a minidystrophin (WO93/06223), tumour suppressor genes: p53, Rb, Rap1A, DCC, k-rev, and the like (WO94/24297), genes encoding factors involved in clotting: Factors VII, VIII, IX and the like, suicide genes: thymidine kinase, cytosin deaminase and the like, or all or part of a natural or artificial immunoglobulin (Fab, ScFv, and the like, WO94/29446), and the like. The gene of interest may also be a gene or an antisense sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences may, for example, be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in Patent EP 140 308. The gene of interest may also be a gene encoding an antigenic peptide, capable of generating an immune response, for the production of vaccines. It may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudorabies virus, or specific for tumours (EP 259 212). The gene may be any DNA (gDNA, CDNA and the like) encoding a product of interest, potentially including the appropriate expression signals (promoter, terminator and the like).
These viruses may be used in vitro for the production of these recombinant proteins. They may also be used, still in vitro, to study the mechanism of action of these proteins or to study the regulation of the expression of genes or the activity of promoters. They may also be used in vivo, for the creation of animal models or of transgenic animals. They may also be used for the transfer and expression of genes in vivo, in animals or man, in gene or cell therapy procedures.
The present application will be described in greater detail with the aid of the following examples which should be considered as illustrative and nonlimiting.